Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation.

Glycosyltransferases are nature's versatile tools to tailor the functionalities of proteins, carbohydrates, lipids, and small molecules by transferring sugars. Prominent substrates are hydroxycoumarins such as scopoletin, which serve as natural plant protection agents. Similarly, C13-apocarotenoids, which are oxidative degradation products of carotenoids/xanthophylls, protect plants by repelling pests and attracting pest predators. We show that C13-apocarotenoids interact with the plant glycosyltransferase NbUGT72AY1 and induce conformational changes in the enzyme catalytic center ultimately reducing its inherent UDP-α-d-glucose glucohydrolase activity and increasing its catalytic activity for productive hydroxycoumarin substrates. By contrast, C13-apocarotenoids show no effect on the catalytic activity toward monolignol lignin precursors, which are competitive substrates. In vivo studies in tobacco plants (Nicotiana benthamiana) confirmed increased glycosylation activity upon apocarotenoid supplementation. Thus, hydroxycoumarins and apocarotenoids represent specialized damage-associated molecular patterns, as they each provide precise information about the plant compartments damaged by pathogen attack. The molecular basis for the C13-apocarotenoid-mediated interplay of two plant protective mechanisms and their function as allosteric enhancers opens up potential applications of the natural products in agriculture and pharmaceutical industry.

Amplification of early drought responses caused by volatile cues emitted from neighboring plants.

Plants have developed sophisticated mechanisms to survive in dynamic environments. Plants can communicate via volatile organic compounds (VOCs) to warn neighboring plants of threats. In most cases, VOCs act as positive regulators of plant defense. However, the communication and role of volatiles in response to drought stress are poorly understood. Here, we showed that tea plants release numerous VOCs. Among them, methyl salicylate (MeSA), benzyl alcohol, and phenethyl alcohol markedly increased under drought stress. Interestingly, further experiments revealed that drought-induced MeSA lowered the abscisic acid (ABA) content in neighboring plants by reducing 9-cis-epoxycarotenoid dioxygenase (NCED) gene expression, resulting in inhibition of stomatal closure and ultimately decreasing early drought tolerance in neighboring plants. Exogenous application of ABA reduced the wilting of tea plants caused by MeSA exposure. Exposure of Nicotiana benthamiana to MeSA also led to severe wilting, indicating that the ability of drought-induced MeSA to reduce early drought tolerance in neighboring plants may be conserved in other plant species. Taken together, these results provide evidence that drought-induced volatiles can reduce early drought tolerance in neighboring plants and lay a novel theoretical foundation for optimizing plant density and spacing.

Six Uridine-Diphosphate Glycosyltransferases Catalyze the Glycosylation of Bioactive C13-Apocarotenols.

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products that perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains poorly understood. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected based on their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and peppermint (Mentha × piperita), as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the glucosylation of C13-apocarotenols, where Glc is bound either to the cyclohexene ring or the butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C), and 3-hydroxy-7,8-dihydro-ß-ionol, whereas a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analysis of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization, and the associated maintenance of carotenoid homeostasis.

Tiered approach for the identification of Mal d 1 reduced, well tolerated apple genotypes.

A rising proportion of the world population suffers from food-related allergies, including incompatibilities to apples. Although several allergenic proteins have been found in apples, the most important proteins that cause allergic reactions to apples in Central-Northern Europe, and North America are the Mal d 1 proteins, which are homologues of the birch pollen allergen Bet v 1. As the demand for hypoallergenic fruits is constantly increasing, we selected apple genotypes with a low total content of Mal d 1 by enzyme-linked immunosorbent assay analysis from segregating populations and tested the tolerability of these fruits through a human provocation study. This tiered approach, which exploited the natural diversity of apples, led to the identification of fruits, which were tolerated by allergic patients. In addition, we found a significant correlation (coefficient >0.76) between the total Mal d 1 content and flavan-3-ol amount and show that the isoform composition of the Mal d 1 proteins, which was determined by LC-MS/MS has a decisive effect on the tolerability of apple genotypes. The approach presented can be applied to other types of fruit and to other allergenic proteins. Therefore, the strategy can be used to reduce the allergen content of other plant foods, thereby improving food safety for allergy subjects.

UGT74AF3 enzymes specifically catalyze the glucosylation of 4-hydroxy-2,5-dimethylfuran-3(2H)-one, an important volatile compound in Camellia sinensis.

4-Hydroxy-2,5-dimethylfuran-3(2H)-one (HDMF) is an important odorant in some fruits, and is proposed to play a crucial role in the caramel-like notes of some teas. However, its biosynthesis and metabolism in tea plants are still unknown. Here, HDMF glucoside was unambiguously identified as a native metabolite in tea plants. A novel glucosyltransferase UGT74AF3a and its allelic protein UGT74AF3b specifically catalyzed the glucosylation of HDMF and the commercially important structural homologues 2 (or 5)-ethyl-4-hydroxy-5 (or 2)-methylfuran-3(2H)-one (EHMF) and 4-hydroxy-5-methylfuran-3(2H)-one (HMF) to their corresponding ß-D-glucosides. Site-directed mutagenesis of UGT74AF3b to introduce a single A456V mutation resulted in improved HDMF and EHMF glucosylation activity and affected the sugar donor preference compared with that of the wild-type control enzyme. The accumulation of HDMF glucoside was consistent with the transcript levels of UGT74AF3 in different tea cultivars. In addition, transient UGT74AF3a overexpression in tobacco significantly increased the HDMF glucoside contents, and downregulation of UGT74AF3 transcripts in tea leaves significantly reduced the concentration of HDMF glucoside compared with the levels in the controls. The identification of HDMF glucoside in the tea plant and the discovery of a novel-specific UDP-glucose:HDMF glucosyltransferase in tea plants provide the foundation for improvement of tea flavor and the biotechnological production of HDMF glucoside.

Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.

Glucosylation of the phytoalexin N-feruloyl tyramine modulates the levels of pathogen-responsive metabolites in Nicotiana benthamiana.

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.

Impact of year of harvest, genotype and cultivation method on bioactives and Pru d 1 allergen content in plums.

The present work studied the effect of the year of harvest, the genotype and the cultivation method on the nutritional quality and the allergen content of three plum cultivars. The common quality parameters and the phytochemical content strongly varied with the year and the cultivar, while the system of cultivation had a minor influence. In particular, ascorbic acid greatly decreased in 2016 compared to 2015, while polyphenols were higher in 2016. The health-promoting compounds, and particularly phenolics, were significantly correlated with the antioxidant capacity. Finally, the allergen content was strongly dependent on the content of flavan-3-ols, suggesting that this class of phenolics is determinant in influencing the allergen content in plums. Results showed that the major factor affecting the quality and the concentration of natural metabolites of plum, in addition to the diversity among genotypes, is the year-to-year variation, whereas the system of cultivation plays a marginal role.

Glucosylation of (Z)-3-hexenol informs intraspecies interactions in plants: A case study in Camellia sinensis.

Plants emit a variety of volatiles in response to herbivore attack, and (Z)-3-hexenol and its glycosides have been shown to function as defence compounds. Although the ability to incorporate and convert (Z)-3-hexenol to glycosides is widely conserved in plants, the enzymes responsible for the glycosylation of (Z)-3-hexenol remained unknown until today. In this study, uridine-diphosphate-dependent glycosyltransferase (UGT) candidate genes were selected by correlation analysis and their response to airborne (Z)-3-hexenol, which has been shown to be taken up by the tea plant. The allelic proteins UGT85A53-1 and UGT85A53-2 showed the highest activity towards (Z)-3-hexenol and are distinct from UGT85A53-3, which displayed a similar catalytic efficiency for (Z)-3-hexenol and nerol. A single amino acid exchange E59D enhanced the activity towards (Z)-3-hexenol, whereas a L445M mutation reduced the catalytic activity towards all substrates tested. Transient overexpression of CsUGT85A53-1 in tobacco significantly increased the level of (Z)-3-hexenyl glucoside. The functional characterization of CsUGT85A53 as a (Z)-3-hexenol UGT not only provides the foundation for the biotechnological production of (Z)-3-hexenyl glucoside but also delivers insights for the development of novel insect pest control strategies in tea plant and might be generally applicable to other plants.

Constitutive Polyphenols in Blades and Veins of Grapevine ( Vitis vinifera L.) Healthy Leaves.

Despite the economic importance and the diffusion of grapevine cultivation worldwide, little is known about leaf chemical composition. We characterized the phenolic composition of Nebbiolo, Barbera, Pinot noir, Cabernet Sauvignon, Grenache, and Shiraz ( Vitis vinifera L.) healthy leaves (separating blades and veins) during the season. Quantitative and qualitative differences were found between leaf sectors and among genotypes. In healthy grapevine leaves, anthocyanins, dihydromyricetin-rhamnoside, hexosides of dihydroquercetin, and dihydrokaempferol exclusively accumulated in veins. Astilbin was the only flavanonol detected in blades and the prevalent flavanonol in veins. Barbera distinguished for the lowest proanthocyanidin and the highest hydroxycinnamate content, and Pinot noir for the absence of acylated-anthocyanins. Nebbiolo, and Cabernet Sauvignon displayed a high concentration of epigallocatechin gallate in veins. Nebbiolo leaves showed the highest concentrations of flavanonols and the widest profile differentiation. Knowledge derived from the present work is a contribution to find out leaf polyphenol potential as a part of grapevine defense mechanisms and to dissect genotype-related susceptibility to pathogens; moreover, it represents a starting point for future deepening about grapevine and vineyard byproducts as a source of bioactive phenolic compounds.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

Lead Development of Thiazolylsulfonamides with Carbonic Anhydrase Inhibitory Action.

A series of congeners structurally related to pritelivir, N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide, a helicase-primase inhibitor for the treatment of herpes simplex virus infections, was prepared. The synthesized primary and secondary sulfonamides were investigated as inhibitors of six physiologically and pharmacologically relevant human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic enzymes hCA I and II, the mitochondrial ones hCA VA and VB, and the transmembrane, tumor associated hCA IX and XII. Low nanomolar inhibition KI values were detected for all of them, with a very interesting and well-defined structure-activity relationship. As many CAs are involved in serious pathologies, among which are cancer, obesity, epilepsy, glaucoma, etc., sulfonamide inhibitors as those reported here may be of interest as drug candidates. Furthermore, pritelivir itself is an effective inhibitor of some CAs, also inhibiting whole blood enzymes from several mammalian species, which may be a favorable pharmacokinetic feature of the drug which can be transported throughout the body bound to blood CA I and II.

Early metabolic and transcriptional variations in fruit of natural white-fruited Fragaria vesca genotypes.

Strawberry fruits (Fragaria vesca) are valued for their sweet fruity flavor, juicy texture, and characteristic red color caused by anthocyanin pigments. To gain a deeper insight into the regulation of anthocyanin biosynthesis, we performed comparative metabolite profiling and transcriptome analyses of one red-fruited and two natural white-fruited strawberry varieties in two tissues and three ripening stages. Developing fruit of the three genotypes showed a distinctive pattern of polyphenol accumulation already in green receptacle and achenes. Global analysis of the transcriptomes revealed that the ripening process in the white-fruited varieties is already affected at an early developmental stage. Key polyphenol genes showed considerably lower transcript levels in the receptacle and achenes of both white genotypes, compared to the red genotype. The expression of the anthocyanidin glucosyltransferase gene and a glutathione S-transferase, putatively involved in the vacuolar transport of the anthocyanins, seemed to be critical for anthocyanin formation. A bHLH transcription factor is among the differentially expressed genes as well. Furthermore, genes associated with flavor formation and fruit softening appear to be coordinately regulated and seem to interact with the polyphenol biosynthesis pathway. This study provides new information about polyphenol biosynthesis regulators in strawberry, and reveals genes unknown to affect anthocyanin formation.

RNAi-mediated endogene silencing in strawberry fruit: detection of primary and secondary siRNAs by deep sequencing.

RNA interference (RNAi) has been exploited as a reverse genetic tool for functional genomics in the nonmodel species strawberry (Fragaria × ananassa) since 2006. Here, we analysed for the first time different but overlapping nucleotide sections (>200 nt) of two endogenous genes, FaCHS (chalcone synthase) and FaOMT (O-methyltransferase), as inducer sequences and a transitive vector system to compare their gene silencing efficiencies. In total, ten vectors were assembled each containing the nucleotide sequence of one fragment in sense and corresponding antisense orientation separated by an intron (inverted hairpin construct, ihp). All sequence fragments along the full lengths of both target genes resulted in a significant down-regulation of the respective gene expression and related metabolite levels. Quantitative PCR data and successful application of a transitive vector system coinciding with a phenotypic change suggested propagation of the silencing signal. The spreading of the signal in strawberry fruit in the 3' direction was shown for the first time by the detection of secondary small interfering RNAs (siRNAs) outside of the primary targets by deep sequencing. Down-regulation of endogenes by the transitive method was less effective than silencing by ihp constructs probably because the numbers of primary siRNAs exceeded the quantity of secondary siRNAs by three orders of magnitude. Besides, we observed consistent hotspots of primary and secondary siRNA formation along the target sequence which fall within a distance of less than 200 nt. Thus, ihp vectors seem to be superior over the transitive vector system for functional genomics in strawberry fruit.

Metabolic engineering of apple by overexpression of the MdMyb10 gene.

Flavonoids are low-molecular-weight phenolic compounds that are widely distributed in the plant kingdom. They have different roles in plant resistance to biotic and abiotic stresses. The transcription factor gene MdMyb10 (Gene Bank: DQ267896) was introduced into two apple (Malus domestica Borkh.) cultivars i.e. 'Holsteiner Cox (HC)' and 'Gala' via Agrobacterium-mediated transformation. The regenerated shoots were selected on kanamycin containing media. The presence of additional MdMyb10 gene in putative shoots was confirmed by PCR, RT-PCR and Southern blotting. Expression level of introduced MdMyb10 gene was analyzed by quantitative real time PCR. The results confirmed a dramatic increase in overexpression of MdMyb10 in the transgenic plants, up to 1261 and 847-folds for cultivars Holsteiner Cox and Gala, respectively compared to non-transformed negative control plants. HPLC-MS was used to determine the levels of different flavonoid compounds in both non-transgenic and transgenic plants. In MdMyb10 'HC' transgenic plants, some of the polyphenols analyzed were enhanced while others were reduced in comparison to their levels in the non-transgenic plants. On the other hand, all of the analyzed polyphenol classes were induced in MdMyb10 'Gala' transgenic plants in comparison to their levels in the non-transgenic plants. In the present study, the flavonoid pathway was successfully modified in apple by overexpressing the MdMyb10 transcription factor to validate the hypothesis of increased effect on plant disease resistance.

Formation of ß-glucogallin, the precursor of ellagic acid in strawberry and raspberry.

Ellagic acid/ellagitannins are plant polyphenolic antioxidants that are synthesized from gallic acid and have been associated with a reduced risk of cancer and cardiovascular diseases. Here, we report the identification and characterization of five glycosyltransferases (GTs) from two genera of the Rosaceae family (Fragaria and Rubus; F. × ananassa FaGT2*, FaGT2, FaGT5, F. vesca FvGT2, and R. idaeus RiGT2) that catalyze the formation of 1-O-galloyl-ß-D-glucopyranose (ß-glucogallin) the precursor of ellagitannin biosynthesis. The enzymes showed substrate promiscuity as they formed glucose esters of a variety of (hydroxyl)benzoic and (hydroxyl)cinnamic acids. Determination of kinetic values and site-directed mutagenesis revealed amino acids that affected substrate preference and catalytic activity. Green immature strawberry fruits were identified as the main source of gallic acid, ß-glucogallin, and ellagic acid in accordance with the highest GT2 gene expression levels. Injection of isotopically labeled gallic acid into green fruits of stable transgenic antisense FaGT2 strawberry plants clearly confirmed the in planta function. Our results indicate that GT2 enzymes might contribute to the production of ellagic acid/ellagitannins in strawberry and raspberry, and are useful to develop strawberry fruit with additional health benefits and for the biotechnological production of bioactive polyphenols.

Genetic dissection of the (poly)phenol profile of diploid strawberry (Fragaria vesca) fruits using a NIL collection.

Over the last few years, diploid strawberry (Fragaria vesca) has been recognized as a model species for applied research of cultivated strawberry (Fragaria × ananassa) that is one of the most economically important crops. Berries, particularly strawberries, are known for their high antioxidant capacity due to a high concentration of (poly) phenolic compounds. Studies have already characterized the phenolic composition of fruits from sets of cultivated strawberries but the quantification of phenolics in a Fragaria mapping population has not been reported, yet. The metabolite profiling of a F. vesca near isogenic line (NIL) collection by LC-MS allowed the unambiguous identification of 22 (poly)-phenols, including anthocyanins, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acid derivatives, and ellagic acid in the diploid strawberry fruit. The variability in the collection revealed that the genetic factor was more decisive than the environmental factor for the accumulation of 18 of the 24 compounds. Genotyping the NIL collection with the Axiom® IStraw90® SNPs array, we were able to map 76 stable QTLs controlling accumulation of the (poly)-phenolic compounds. They provide a powerful new tool to characterise candidate genes to increase the antioxidant capacity of fruits and produce healthier strawberries for consumers.

Bioactive C17-Polyacetylenes in Carrots (Daucus carota L.): Current Knowledge and Future Perspectives.

C17-polyacetylenes (PAs) are a prominent group of oxylipins and are primarily produced by plants of the families Apiaceae, Araliaceae, and Asteraceae, respectively. Recent studies on the biological activity of polyacetylenes have indicated their potential to improve human health due to anticancer, antifungal, antibacterial, anti-inflammatory, and serotogenic effects. These findings suggest targeting vegetables with elevated levels of bisacetylenic oxylipins, such as falcarinol, by breeding studies. Due to the abundant availability, high diversity of cultivars, worldwide experience, and high agricultural yields, in particular, carrot (Daucus carota L.) genotypes are a very promising target vegetable. This paper provides a review on falcarinol-type C17-polyacetylenes in carrots and a perspective on their potential as a future contributor to improving human health and well-being.

FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.).

The strawberry (Fragaria × ananassa) is one of the most preferred fresh fruit worldwide, accumulates numerous flavonoids but has limited shelf life due to excessive tissue softening caused by cell wall degradation. Since lignin is one of the polymers that strengthen plant cell walls and might contribute to some extent to fruit firmness monolignol biosynthesis was studied in strawberry fruit. Cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and a peroxidase (POD27) gene were strongly expressed in red, ripe fruit whereas a second POD gene was primarily expressed in green, immature fruit. Moreover, FaPOD27 transcripts were strongly and constitutively induced in fruits exposed to Agrobacterium infection. Gene expression levels and enzymatic activities of FaCCR and FaCAD were efficiently suppressed through RNAi in FaCCR- and FaCAD-silenced strawberries. Besides, significantly elevated FaPOD transcript levels were detected after agroinfiltration of pBI-FaPOD constructs in fruits. At the same time, levels of G-monomers were considerably reduced in FaCCR-silenced fruits whereas the proportion of both G- and S-monomers decisively decreased in FaCAD-silenced and pBI-FaPOD fruits. Development, firmness, and lignin level of the treated fruits were similar to pBI-intron control fruits, presumably attributed to increased expression levels of FaPOD27 upon agroinfiltration. Additionally, enhanced firmness, accompanied with elevated lignin levels, was revealed in chalcone synthase-deficient fruits (CHS(-)), independent of down- or up-regulation of individual and combined FaCCR. FaCAD, and FaPOD genes by agroinfiltration, when compared to CHS(-)/pBI-intron control fruits. These approaches provide further insight into the genetic control of flavonoid and lignin synthesis in strawberries. The results suggest that FaPOD27 is a key gene for lignin biosynthesis in strawberry fruit and thus to improving the firmness of strawberries.